Mammalian cells employ a variety of innate immune mechanisms to recognize and eliminate invading microorganisms, including autophagy of intracellular particles and pro-inflammatory cytokine mediated recruitment of inflammatory cells to the site of infection. Within minutes of Shigella spp. or Salmonella spp. entry into cells, autophagy is activated and inflammasome receptors and adaptors are recruited around intracellular bacteria. A substantial subpopulation of intracellular bacteria avoids association with autophagy markers. How this subpopulation of bacteria evades recognition by these markers has been unclear. Our preliminary data suggest a model in which S. flexneri actively protects itself from autophagy markers. We find that the subpopulation of S. flexneri not associated with certain autophagy markers is universally associated with the host multifunctional protein Toca-1. Based on our findings and on published data, we hypothesize that in the setting of vacuolar membrane remnants, which normally induce innate immune responses, Toca-1 serves as a scaffold that (1) is anchored to membrane fragments through IcsB and IpaB, and (2) coordinates repression of autophagy and restriction of pro-inflammatory cytokine production. Specifically, we hypothesize that (i) IpaB, IcsB, OspC2/3 proteins, and Toca-1 form a complex associated with vacuolar membrane remnants; (ii) the complex assembles on IpaB that has been inserted into the plasma membrane soon after contact of bacteria with the membrane; and, (iii) the IpaB-IcsB-Toca-1-OspC2/3 protein complex per se interferes with pro- inflammatory cytokine production by promoting OspC3-mediated inhibition of caspase activation. The exploratory investigations proposed in this R21 application will test aspects of this hypothesis. Aim 1: Investigate whether IcsB, OspC2/3 proteins, IpgD, and/or Toca-1 form a complex associated with vacuolar membrane, and if they do, characterize how. Aim 2: Investigate whether the IpaB-IcsB-Toca-1-OspC2/3 protein complex promotes interference with caspase activation. The proposed investigations are highly likely to lead to new insights into mechanisms of S. flexneri manipulation of innate immune responses early during infection. The results obtained from these investigations are likely to have important implications for infection by all intracellular microorganisms and for modulation of innate immune responses more generally.